Chia seeds as a source of natural lipid antioxidants

Chia (Salvia sp) seeds were investigated as a source of natural lipid antioxidants. Methanolic and aqueous extracts of defatted chia seeds possessed potent antioxidant activity. Analysis of 2 batches of chia-seed oils demonstrated marked difference in the fatty acid composition of the oils. In both batches, the oils had high concentrations of polyunsaturated fatty acids. The major antioxidant activity in the nonhydrolyzed extract was caused by flavonol glycosides, chlorogenic acid (7.1 × 10⁻⁴ mol/kg of seed) and caffeic acid (6.6 × 10⁻³ m/kg). Major antioxidants of the hydrolyzed extracts were flavonol aglycones/kaempferol (1.1 × 10⁻³ m/kg), quercetin (2.0 × 10⁻⁴ m/kg) and myricetin (3.1 × 10⁻³ m/kg); and caffeic acid (1.35 × 10⁻² m/kg). Two methods were used to measure antioxidant activities. Both were based on measuring bleaching ofβ-carotene in the coupled oxidation ofβ-carotene and linoleic acid in the presence of added antioxidants.     

Arachidonic acid autoxidation in an aqueous media effect of α-tocopherol,cysteine and nucleic acids

The autoxidation of arachidonic acid dispersed in aqueous media was evaluated simultaneously with and without different agents, e.g., α-tocopherol at different concentrations, cysteine, DNA and RNA. The autoxidation rate of arachidonic acid was evaluated by quantitative gas liquid chromatography (GLC) determination of the unoxidized acid and by spectrophotometric measurement of conjugated dienes. α-Tocopherol exhibited a prooxidant activity at concentrations of 1.25 × 10⁻⁴ M and 1.25 × 10⁻⁵ M and a weak antioxidant activity at a concentration of 1.25 × 10⁻⁶ M. Cysteine showed antioxidant activity and greatly reduced the prooxidant activity of α-tocopherol. DNA and RNA had no effect in either case. α-Tocopherol oxidation was followed by high pressure liquid chromatography (HPLC). The prooxidant effect was accompanied by a rapid oxidation of α-tocopherol, except in the presence of cysteine, which prevented the oxidation of α-tocopherol.     

Kinetic study of the reaction between tocopheroxyl radical and unsaturated fatty acid esters in benzene

A kinetic study of the reaction between a tocopheroxyl radical and unsaturated fatty acid esters has been undertaken. The rates of allylic hydrogen abstraction from various unsaturated fatty acid esters (ethyl oleate2, ethyl linoleate3, ethyl linolenate4, and ethyl arachidonate5) by the tocopheroxyl radical (5,7-diisopropyltocopheroxyl6) in benzene have been determined spectrophotometrically. The second-order rate constants, k₃, obtained are 1.04×10⁻⁵ M⁻¹s⁻¹ for2, 1.82×10⁻² M⁻¹s⁻¹ for3, 3.84×10⁻² M⁻¹s⁻¹ for4, and 4.83×10⁻² M⁻¹s⁻¹ for5 at 25.0°C. Thus, the rate constants, k_abstr/H, given on an available hydrogen basis are k₃/4=2.60×10⁻⁶ M⁻¹s⁻¹ for2, k₃/2=9.10×10⁻³ M⁻¹s⁻¹ for3, k₃/4=9.60×10⁻³ M⁻¹s⁻¹ for4, and k₃/6=8.05×10⁻³ M⁻¹s⁻¹ for5. The k_abstr/H values obtained for the polyunsaturated fatty acid esters3,4, and5 containing H-atoms activated by two π-electron systems are similar to each other, and are about three orders of magnitude higher than that for the ethyl oleate2 containing H-atoms activated by a single π-system. From these results, it is suggested that the prooxidant effect of α-tocopherol in edible oils and fats may be induced by the above hydrogen abstraction reaction.     

Kinetic Study of the Prooxidant Effect of α-Tocopherol. Hydrogen Abstraction from Lipids by α-Tocopheroxyl Radical

A kinetic study of the prooxidant effect of α-tocopherol was performed. The rates of allylic hydrogen abstraction from various unsaturated fatty acid esters (ethyl stearate 1, ethyl oleate 2, ethyl linoleate 3, ethyl linolenate 4, and ethyl arachidonate 5) by α-tocopheroxyl radical in toluene were determined, using a double-mixing stopped-flow spectrophotometer. The second-order rate constants (k _p) obtained are <1 × 10⁻² M⁻¹ s⁻¹ for 1, 1.90 × 10⁻² M⁻¹ s⁻¹ for 2, 8.33 × 10⁻² M⁻¹ s⁻¹ for 3, 1.92 × 10⁻¹ M⁻¹ s⁻¹ for 4, and 2.43 × 10⁻¹ M⁻¹ s⁻¹ for 5 at 25.0 °C. Fatty acid esters 3, 4, and 5 contain two, four, and six –CH₂– hydrogen atoms activated by two π-electron systems (–C=C–CH₂–C=C–). On the other hand, fatty acid ester 2 has four –CH₂– hydrogen atoms activated by a single π-electron system (–CH₂–C=C–CH₂–). Thus, the rate constants, k _abstr/H, given on an available hydrogen basis are k _p/4 = 4.75 × 10⁻³ M⁻¹ s⁻¹ for 2, k _p/2 = 4.16 × 10⁻² M⁻¹ s⁻¹ for 3, k _p/4 = 4.79 × 10⁻² M⁻¹ s⁻¹ for 4, and k _p/6 = 4.05 × 10⁻² M⁻¹ s⁻¹ for 5. The k _abstr/H values obtained for 3, 4, and 5 are similar to each other, and are by about one order of magnitude higher than that for 2. From these results, it is suggested that the prooxidant effect of α-tocopherol in edible oils, fats, and low-density lipoproteins may be induced by the above hydrogen abstraction reaction.     

α-Tocopherol prooxidant effect and malondialdehyde production

α-Tocopherol at high concentration (1.25×10⁻⁴M) exhibited a prooxidant effect during autoxidation of linolenic and arachidonic acids. This prooxidant activity involved a significant increase of the conjugated diene level, especially with linolenic acid. High performance liquid chromatographic evaluation of malondialdehyde, a by-product of the hydroperoxides of polyunsaturated fatty acids, showed that malondialdehyde was not increased during prooxidant effect ofα-tocopherol. Thus, malondialdehyde does not seem to be a good indicator for the manifestation of the prooxidant activity ofα-tocopherol.     

Evaluation of antioxidant activity: II. application of a heme- Catalyzed system

A study was made of the inhibitory effect of the antioxidants propyl gallate,tert-butylated hydroxyanisol, α-tocopherol and ethoxyquin on the hemoglobin-catalyzed oxygenation of linoleic acid. The concentration of unchanged fatty acid after varying incubation periods and at varying concentrations of antioxidant was measured by gas chromatography. The effect of the antioxidants is compared with results obtained previously from the lipoxygenase-catalyzed oxidation of a linoleic acid emulsion. It is concluded that all 4 antioxidants are good inhibitors of fatty acid oxidation cata-lyzed by hemoglobin.     

Flavonoids as stabilizers of fish oil: An alternative to synthetic antioxidants

The antioxidant activities against fish oil oxidation of six commercially available flavonoids and of five flavonoids purified from two Chilean native plants were compared to those ofdl-α-tocopherol and of two synthetic antioxidants, butylated hydroxytoluene and butylated hydroxyanisole. Among the commercial flavonoids, catechin, morin and quercetin showed a higher activity when fish oil oxidation (either spontaneous or Fe²⁺-induced) was assessed from the formation of peroxides or thiobarbituric acid-reactive substances. Among the native flavonoids, the 5,3′,4′-trihydroxy-7-methoxy flavanone (designated as Pt-2) showed the highest antioxidant activity. Mixtures of quercetin or of Pt-2 withdl-α-tocopherol produced better inhibitory effects when compared to that of each substance assayed by itself. Also, when Pt-2 and quercetin were assayed in combination (0.3 g/kg oil and 0.7 g/kg oil, respectively), a synergistic antioxidant effect was observed. Results indicate that several flavonoids could be used as natural antioxidants as a means to replace those synthetic antioxidants, the use of which has been questioned.     

Radiolytic resistance of DL-α-tocopherol in lipid systems with different degrees of unsaturation

The radiolytic resistance of the DL-α-tocopherol irradiated by low (10⁵rad), medium (10⁶rad) and high (10⁷rad) doses of gamma rays at a molar ratio of 1:1, 1:1 × 10⁻² and 1:1 × 10⁻³ mole in methyl laurate, methyl oleate, methyl linoleate, methyl linolenate and benzene (chosen as solvent media) has been studied. Under the experimental conditions stated, it has been established that, contrary to ordinary autoxidation, the unsaturated lipid systems exert a progressive, protective effect on DL-α-tocopherol as the number of double bonds increases. When the DL-α-tocopherol was in a pure state, for example in benzene and in methyl esters of the fatty acids at a molar ratio 1:1, no effect of ionizing radiation was detected.     

Stabilization of seal blubber and menhaden oils with green tea catechins

Catechins, namely (−)epicatechin (EC), (−)epigallocatechin (EGC), (−)epicatechin gallate (ECG) and (−)epigallocatechin gallate (EGCG), were isolated from commercial Chinese green leaves. The antioxidant activity of isolated catechins was compared with those of α-tocopherol, butylated hydroxyanisole (BHA), butylated hydroxytolene (BHT) andtert-butylhydroquinone (TBHQ), all at 200 ppm, in refined, bleached and deodorized seal blubber oil and menhaden oil. The study was carried out under Schaal oven test conditions at 65°C over a 144-h period, except for weight gain measurements, which were continued for up to 200 h. Progression of oxidation was monitored by measuring changes in weight gain and values of peroxide, conjugated diene, and 2-thiobarbituric acid-reactive substances. Oils treated with tea catechins showed excellent oxidative stability as compared with samples that contained commonly used antioxidants, such as α-tocopherol, BHA, BHT, and TBHQ. The potency of catechins in prevention of oxidation of marine oils was in the decreasing order of ECG> EGCG> EGC> EC; ECG was slightly more effective than TBHQ in systems studied.     

Sensory Thresholds of Selected Phenolic Constituents from Thyme and their Antioxidant Potential in Sunflower Oil

The odor detection thresholds of carvacrol (5-isopropyl-2-methyl-phenol), thymol (2-isopropyl-5-methyl-phenol) and p-cymene 2,3-diol (2,3-dihydroxy-4-isopropyl-1-methyl-benzene) in sunflower oil, determined by the three-alternative, forced-choice procedure, were 30.97, 124 and 794.33 mg kg⁻¹, respectively. Sunflower oil containing 13, 70, or 335 mg kg⁻¹ of carvacrol, thymol or p-cymene 2,3-diol, respectively, was judged to be similar (P < 0.01) in taste and odor to its antioxidant-free counterpart. The rate constant of sunflower oil oxidation, measured from the increase in peroxide value during storage at 25 °C, was 9.2 × 10⁻⁹ mol kg⁻¹ s⁻¹ while the rate constants were 9.3 × 10⁻⁹, 9.8 × 10⁻⁹, and 4.3 × 10⁻⁹ mol kg⁻¹ s⁻¹ in the presence of 13 mg kg⁻¹ carvacrol, 70 mg kg⁻¹ thymol, and 335 mg kg⁻¹ p-cymene 2,3-diol, respectively. At a level of 335 mg kg⁻¹, p-cymene 2,3-diol did not impart flavor taints and effected a 46.7% reduction in the rate of oxidation of sunflower oil. These findings indicate that the diphenolic p-cymene 2,3-diol could potentially replace synthetic antioxidants and is a valuable addition to the antioxidants used by the food industry in its quest to meet consumer demands for synthetic-additives-free and ‘natural’ foods.     

Voltammetric behavior and the determination of quercetin at a flowerlike Co<Subscript>3</Subscript>O<Subscript>4</Subscript> nanoparticles modified glassy carbon electrode

Flowerlike Co₃O₄ nanoparticles were used as a modifier on the glassy carbon electrode to fabricate a quercetin (Qu) sensor. The morphology and crystallinity of the prepared Co₃O₄ material were investigated by scanning electron microscopy and X-ray diffraction. Electrochemical behavior of Qu at the sensor was studied by cyclic voltammetry and semi-derivative voltammetry. Results suggested that the modified electrode exhibited a strong electrocatalytic activity toward the redox of Qu. The electron transfer coefficient (α), the number of electron transfer (n), and the diffusion coefficient (D) of Qu at the sensor were calculated. Under the optimum conditions, the catalytic peak currents of Qu were linearly dependent on the concentrations of Qu in the range from 5.0 × 10⁻⁷ to 3.3 × 10⁻⁴ M, with a detection limit of 1.0 × 10⁻⁷ M. This proposed method was successfully applied to determine the quercetin concentration in Ginkgo leaf tablet and human urine samples.     

α-tocopherol oxidation mediated by superoxide anion (O 2 − ). II. Identification of the stable α-tocopherol oxidation products

The present paper describes the identification of two stable end products of α-tocopherol oxidation that were previously detected among the products of the reaction of α-tocopherol with superoxide anion (O ₂ ⁻ ) under aprotic conditions. One compound, previously designated compound A, was identified astrans-7-hydroxy-trans-8,8a-epoxy-α-tocopherone, and the other, designated compound B, was identified ascis-7-hydroxy-cis-8,8a-epoxy-α-tocopherone. It was also observed that under protic conditions (10% water in acetonitrile) the reaction of α-tocopherol with O ₂ ⁻ did not produce compounds A and B, but rather α-tocopheryl quinone, α-tocopherol dimer, α-tocopherol dihydroxy dimer, and the previously designated compound C. Compound C was identified in the present study as α-tocopheryl-quinone-2,3-epoxide.     

Rate constants for quenching singlet oxygen and activities for inhibiting lipid peroxidation of carotenoids and α-tocopherol in liposomes

The ¹O₂ quenching rate constants (k _Q ) of α-tocopherol (α-Toc) and carotenoids such as β-carotene, astaxanthin, canthaxanthin, and lycopene in liposomes were determined in light of the localization of their active sites in membranes and the micropolarity of the membrane regions, and compared with those in ethanol solution. The activities of α-Toc and carotenoids in inhibiting ¹O₂-dependent lipid peroxidation (reciprocal of the concentration required for 50% inhibition of lipid peroxidation: [IC₅₀]⁻¹) were also measured in liposomes and ethanol solution and compared with their k _Q values. The k _Q and [IC₅₀]⁻¹ values were also compared in two photosensitizing systems containing Rose bengal (RB) and pyrenedodecanoic acid (PDA), respectively, which generate ¹O₂ at different sites in membranes. The k _Q values of α-Toc were 2.9×10⁸M⁻¹s⁻¹ in ethanol solution and 1.4×10⁷ M⁻¹s⁻¹ (RB system) or 2.5×10⁶ M⁻¹s⁻¹ (PDA system) in liposomes. The relative [IC₅₀]⁻¹ value of α-Toc in liposomes was also five times higher in the RB system than in the PDA-system. In consideration of the local concentration of the OH-group of α-Toc in membranes, the k _Q value of α-Toc in liposomes was recalculated as 3.3×10⁶ M⁻¹s⁻¹ in both the RB and PDA systems. The k _Q values of all the carotenoids tested in two photosensitizing systems were almost the same. The k _Q value of α-Toc in liposomes was 88 times less than in ethanol solution, but those of carotenoids in liposomes were 600–1200 times less than those in ethanol solution. The [IC₅₀]⁻¹ value of α-Toc in liposomes was 19 times less than that in ethanol solution, whereas those of carotenoids in liposomes were 60–170 times less those in ethanol solution. There were no great differences (less than twice) in the k _q and [IC₅₀]⁻¹ values of any carotenoids. The k _Q values of all carotenoids were 40–80 times higher than that of α-Toc in ethanol solution but only six times higher that of α-Toc in liposomes. The [IC₅₀]⁻¹ values of carotenoid were also higher than that of α-Toc in ethanol solution than in liposomes, and these correlated well with the k _Q values.     

Antioxidant activity of Magnolol, honokiol, and related phenolic compounds

The antioxidant activity of 10 Japanese and Chinese crude drugs (Kampo drugs) was determined in vitro. Extract of Magnolia cortex, which had the highest antioxidant activity, contained phenolic compounds magnolol and honokiol. However, inhibitory effects of these compounds on lipid oxidation were weaker than that of α-tocopherol as measured by thiobarbituric acid assay. The structure-activity relationship of phenolic compounds showed that antioxidant activities were in the order 4-allyl-2,6-dimethoxyphenol ≥ p,p′-biphenol > eugenol > 2-allyl-6-methylphenol > honokiol > magnolol > caffeic acid > p-ethylphenol > guaiacol. As expected, these results showed that an electron donor and/or bulky groups at the ortho- or para-position of the phenol were required for inhibition of lipid oxidation. Electron spin resonance spin trapping experiments showed that phenol compounds with an allyl substituent on their aromatic rings directly scavenged superoxide (O ₂ ⁻ ), and that only eugenol trapped hydroxyl radicals. These findings suggest that phenolic compounds that contain allyl groups may be effective antioxidants because of the scavenging ability of O ₂ ⁻ or hydroxyl radical, whereas other phenols, without an allyl moiety such as α-tocopherol, may play a role in the termination of free radical chain reactions.     

Effectiveness of antioxidants in refined,bleached avocado oil

The addition of antioxidants propyl gallate (PG), α-tocopherol and ethoxyquin at a level of 250 ppm to refined, bleached avocado oil resulted in the retardation of the oxidative deterioration of the oil when it was stored in the dark at room temperature, exposed to daylight at room temperature (on the shelf) and at 60 C. The extent of oxidation was followed by measuring the peroxide and anisidine values and oil color. Ethoxyquin and α-tocopherol were relatively ineffective antioxidants, whereas PG greatly improved the stability of avocado oil stored in the dark at 60 C, but not in oil exposed to daylight.     

Cholesterol oxides II. Measurement of the 5,6-epoxides during cholesterol oxidation in aqueous dispersions

Cholesterol in aqueous dispersion with sodium stearate or Triton surfactants was aerated at various pH values at 50 and 80 C. Analysis of the reaction mixtures by TLC during the oxidation produced qualitatively similar patterns regardless of pH or temperature. Major oxidation products observed were 7-ketocholesterol, the isomeric 7-hydroperoxy- and 7-hydroxycholesterols, the isomeric 5,6-epoxycholestanols and 3β,5α,6β-trihydroxycholestanol. The concentrations of 3β,5α,6β-trihydroxycholestanol and an unknown compound increased greatly at the lower pH values. Recovery of the 5,6-epoxide isomers by preparative TLC followed by capillary GC allowed theα- andβ-epoxide isomers to be quantitated. Oxidations at pH 8 and 12 produced increasing amounts of the epoxides with time, without significant changes in theα/β-epoxide ratio. However, oxidations at the acidic pH values of 5.5 and 3 showed large changes in theα/β-epoxide ratio during the oxidation. Measurement of the hydrolysis rates of the 5,6-epoxides at pH 5.5 showed that theβ-epoxide isomer is more labile than theα-isomer by a factor of 2.5. The rate constant for the hydrolysis of theα-epoxide isomer was 5.3 × 10⁻⁷ sec⁻¹ and that of theβ-isomer 13 × 10⁻⁷ sec⁻¹.     

Concentration and stabilization of n-3 polyunsaturated fatty acids from sardine oil

A simple and relatively inexpensive procedure to obtain 90% eicosapentaenoic acid + docosahexaenoic acid concentrates from sardine oil involved a two-step winterization of the oil (10 and 4°C), followed by saponification and selective precipitation of saturated and less unsaturated free fatty acids by an ethanolic solution of urea. Antioxidant effects of butylated hydroxytoluene, dl-α-tocopherol, and two natural antioxidants, quercetin and boldine, added to the concentrate (0.5% wt/vol), were compared in the Rancimat at 60°C. dl-α-Tocopherol was unable to inhibit concentrate oxidation. Butylated hydroxyanisole and butylated hydroxytoluene had induction periods of 1.7–1.8 h compared to the control (1.0 h). A mixture of quercetin + boldine (2:1 w/w) significantly increased the induction period to 4.5 h.     

Antioxidant Activity of Added Phenolic Compounds in Freeze-Dried Microencapsulated Sunflower Oil

This study was aimed at evaluating the effectiveness of phenolic antioxidants with a similar structure but having a different polarity in dried microencapsulated sunflower oil. The antioxidants tested were, on one hand, α-tocopherol and its water soluble analogue, Trolox, and on the other, gallic acid and its ester derivatives, propyl gallate and dodecyl gallate. At a moderate temperature (40 °C), the samples were oxidized under accelerated conditions by using Cu(II) as an oxidation catalyst. The progress of oxidation was followed up over time in the free and encapsulated oil fractions. The peroxide value, the total content of polymers and, when appropriate, the content of α-tocopherol were determined. Quantitative analysis of the total fraction of the non-volatile oxidation products and their distribution in oligomers, dimers and monomers was applied to samples to obtain a complete evaluation of oxidation. Finally, as a complementary measure, the antioxidants were also assessed by direct application of the Rancimat test at 100 °C on the dried microencapsulated oil samples. Results showed that the antioxidants of lower polarity in each series, i.e. tocopherol and dodecyl gallate, were to a great extent the most protective antioxidants. The results obtained by the Rancimat test were consistent with those found during oxidation at moderate temperature. Furthermore, the addition of Cu(II) reduced proportionally the oxidative stability index of the dried microencapsulated samples.     

Autoxidation of methyl linoleate initiated by the ozonide of allylbenzene

Allylbenzene ozonide (ABO), a model for polyunsaturated fatty acid (PUFA) ozonides, initiates the autoxidation of methyl linoleate (18∶2 ME) at 37°C under 760 torr of oxygen. This process is inhibited by d-α-tocopherol (α-T) and 2,6-di-ert-butyl-4-methylphenol (BHT). The autoxidation was followed by the appearance of conjugated diene (CD), as well as by oxygen-uptake. The rates of autoxidation are proportional to the square root of ABO concentration, implying that the usual free radical autoxidation rate law is obeyed. Activation parameters for the thermal decomposition of ABO were determined under N₂ in the presence of radical scavengers and found to be E_a=28.2 ±0.3 kcal mol⁻¹ and log A=13.6±0.2; k_d (37°C) is calculated to be (5.1±0.3)×10⁻⁷ sec⁻¹. Autoxidation data are also reported for ozonides of 18∶2 ME and methyl oleate (18∶1 ME).     

HPLC analysis of phenolic antioxidants,tocopherols and triglycerides

Triglycerides, together with nine synthetic phenolic antioxidants most commonly used to prevent oxidation of edible oils and fats, as well as the natural antioxidants tocopherols andα-tocopherol acetate, were separated by high performance liquid chromatography by means of a reversed phase C₁₈-column and gradient elution with water/acetonitrile/methanol/isopropanol. Besides dilution of the oil with isopropanol/hexane, no further example preparation was required. UV detection was applied. The synthetic antioxidants propyldodecylgallate, octyldodecylgallate, dodecylgallate, 3-tert-butyl-4-hydroxyanisol,tert-butylhydroquinone, 3,5-di-terf-butyl-4-hydroxytoluene, 2,6-di-tert-butyl-4-hydroxymethyl-phenol, 2,4,5-trihydroxybutyrophenone and nordihydro-quaiaretic acid, as well asα- andδ-tocopherol andα-tocopherol acetate were base-line separated;β- andγ-tocopherol, however, eluted together. The triglycerides, detected at λ = 215 nm, were separated according to then-partition number. The absorption at λ = 215 nm revealed saturated and unsaturated triglycerides. The absorption at λ = 280 nm indicated triglycerides with conjugated unsaturation, relating information about refining and heat treatment of the oil. Oxidized unsaturated triglycerides showed absorption at γ = 230 nm. Triglycerides of ricinoleic acid, a hydroxymonoun-saturated acid, gave identical UV spectra. The simultaneous detection of antioxidants and triglycerides may be used to study inhibition effects by antioxidants in oils. To whom correspondence should be addressed.